EP1341897A2 - Systeme d'hybridation in situ de mise en evidence specifique de micro-organismes - Google Patents

Systeme d'hybridation in situ de mise en evidence specifique de micro-organismes

Info

Publication number
EP1341897A2
EP1341897A2 EP01991823A EP01991823A EP1341897A2 EP 1341897 A2 EP1341897 A2 EP 1341897A2 EP 01991823 A EP01991823 A EP 01991823A EP 01991823 A EP01991823 A EP 01991823A EP 1341897 A2 EP1341897 A2 EP 1341897A2
Authority
EP
European Patent Office
Prior art keywords
slide
sample
nucleic acid
hybridization
arrangement according
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Granted
Application number
EP01991823A
Other languages
German (de)
English (en)
Other versions
EP1341897B1 (fr
Inventor
Peter MÜHLHAHN
Jiri Snaidr
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Vermicon AG
Original Assignee
Vermicon AG
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Vermicon AG filed Critical Vermicon AG
Publication of EP1341897A2 publication Critical patent/EP1341897A2/fr
Application granted granted Critical
Publication of EP1341897B1 publication Critical patent/EP1341897B1/fr
Anticipated expiration legal-status Critical
Expired - Lifetime legal-status Critical Current

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6813—Hybridisation assays
    • C12Q1/6841—In situ hybridisation
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
    • C12Q1/689—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms for bacteria

Definitions

  • the invention relates to an in situ hybridization arrangement for the specific detection of microorganisms, a method for the specific detection of microorganisms by in situ hybridization and a K t with which the identification and visualization of microorganisms in a sample is possible.
  • PCR the polymerase chain reaction
  • a specific, characteristic piece of the bacterial genome is amplified using bacteria-specific primers. If the primer finds its destination, a piece of the genetic material multiplies millions of times.
  • a qualitative evaluation can take place. In the simplest case, this leads to the statement that the target sites are present in the examined sample. No further statements are permitted since the target sites can come from a living bacterium, from a dead bacterium or from naked DNA. A differentiation is not possible here.
  • a further development of this technique is quantitative PCR, in which an attempt is made to establish a correlation between the amount of bacteria present and the amount of DNA obtained and amplified.
  • biochemical parameters are also used for bacterial identification: For example, the creation of bacterial profiles based on quinone determinations serves to reproduce an image of the bacterial population that is as distortion-free as possible (Hiraishi, A. 1988. Respiratory quinone profiles as tools for identifying diffemet bacterial populations in activated sludge. J. Gen. Appl. Microbiol. 34: 39-56).
  • this method also depends on the cultivation of individual bacteria, since the quinone profiles of the bacteria in pure culture are required to create the reference database.
  • the determination of the bacterial quinone profiles does not give a real impression of the actual population relationships in the sample.
  • the often large-volume and bulky antibody-fluorescent molecule complex causes problems when entering the target cells.
  • the detection is often too specific.
  • Antibodies which are expensive to produce, specifically often only detect a specific strain of bacteria, but leave other strains of the same type of bacteria undetected. Often, however, it is not necessary to have a strain-specific detection of the bacteria, but rather to detect a type of bacteria or an entire group of bacteria.
  • antibody production is a relatively lengthy and expensive process.
  • the fluorescence-in-situ method offers a unique approach to combine the specificity of molecular biological methods such as PCR with the possibility of bacterial visualization, as made possible by the antibody method.
  • Hybridization FISH; Amann, RI, W. Ludwig, and K.-H. Schleifer. 1995. Phylogenetic identification and in situ detection of individual microbial cells without cultivation. Microbial. Rev. 59: 143-169).
  • Bacterial species, genera or groups can be visualized and identified directly in the sample in a highly specific manner. This method is the only approach that provides a distortion-free representation of the actual in situ conditions of the biocenosis. Even bacteria that have not yet been cultivated and therefore not described can be identified and also visualized directly in the sample.
  • the FISH technique is based on the fact that there are certain molecules in bacterial cells that, due to their vital function, have undergone only little mutation in the course of evolution: the 16S and the 23S ribosomal ribonucleic acid (rRNA). Both are components of the ribosomes, the sites of protein biosynthesis, and due to their ubiquitous distribution, their size, and their structural and functional constancy, they can serve as phylogenetic markers (Woese, CR 1987. Bacterial evolution. Microbiol. Rev. 51: 221-271) , Based on a comparative sequence analysis, phylogenetic relationships can be established based solely on this data. To do this, this sequence data must be aligned.
  • Figure 1 shows the secondary structure model of a 16S rRNA.
  • phylogenetic calculations can be carried out.
  • the use of the latest computer technology makes it possible to carry out large-scale calculations quickly and effectively, and to create large databases that contain the alignment sequences of the 16S rRNA and 23S rRNA. Thanks to the quick access to this data material newly obtained sequences can be analyzed phylogenetically in a short time.
  • These rRNA databases can be used to construct specific gene probes. Here, all available rRNA sequences are compared with each other and probes designed for specific sequence sites that specifically record a bacterial species, genus or group.
  • FISH fluorescence in situ hybridization
  • these gene probes which are complementary to a specific region on the ribosomal target sequence, are smuggled into the cell.
  • the gene probes are usually small, 16-20 base long, single-stranded deoxyribonucleic acid pieces and are directed against a target region, which is typical for a type or group of bacteria. If the fluorescence-labeled gene probe finds its target sequence in a bacterial cell, it binds to it and the cells can be detected in the fluorescence microscope due to their fluorescence.
  • Figure 2 illustrates the process of in situ hybridization.
  • Bacterial population of a sample can be made visible, the FISH technique allows detection of up to 100% of the total bacterial population in many samples.
  • the active portion of a population can be determined by the ratio of the probe directed against all bacteria and an unspecific cell staining.
  • the bacteria are made visible directly at the place where they act (in situ). Possible interactions between different bacterial populations can thus be recognized and analyzed.
  • the detection of bacteria using the FISH technique is much faster than using cultivation. If the identification of bacteria by cultivation often takes several days, only a few hours pass from sampling to bacterial identification even at the species level using the FISH technique.
  • the specificity of gene probes can be chosen freely as desired. Individual species can be detected with a probe just as easily as entire genera or groups of bacteria.
  • bacterial species or entire bacterial populations can be precisely quantified directly in the sample.
  • the detour via cultivation and the associated insufficient quantification need not be taken.
  • a bacterium occurring in a sample is examined for its taxonomy, the top-to-bottom approach is used.
  • the bacterial sample is analyzed at the beginning of the investigation with gene probes that are as broad as possible, ie, not very specific and only cover entire groups of bacteria. A gradual increase in the specificity of the probes used finally allows the identification of the unknown bacterium.
  • the FISH technique is therefore an outstanding tool for quickly and extremely specifically detecting bacteria directly in a sample. In contrast to cultivation methods, it is a direct method and allows compared to modern ones
  • the FISH analysis is always carried out on a slide, since during the evaluation the bacteria are visualized, ie made visible, by irradiation with high-energy light.
  • the composition of the individual solutions such as hybridization buffer or hybridization solution and wash buffer or wash solution is well known to the person skilled in the art and is e.g. in Snaidr et. al, 1997 (J. Snaidr, R. Amann, I. Huber, W. Ludwig, and K.-H. Schleifer. 1997. Phylogenetic analysis and in situ identification of bacteria in activated sludge. Appl. Env. Microb. 63: 7, 2884-2896).
  • Carrying out the FISH for the analysis of microorganisms on a slide usually comprises the steps shown below.
  • Ethanol series The slide is immersed in a 50%, 70% and 100% ethanol solution one after the other. 6. Apply a hybridization solution to the recess containing the microorganisms. 7. Apply a probe solution to the same recess.
  • the slide is placed horizontally on the pulp in the humid chamber.
  • the wet chamber is placed in an incubation oven and incubated for 1-2 hours.
  • the moist chamber is opened, the slide is removed and briefly rinsed with distilled water. 12. The damp chamber is discarded.
  • a wash buffer solution is introduced into a new plastic tube.
  • the rinsed slide is inserted into the plastic tube filled with the wash solution.
  • the slide is incubated in the plastic tube in an incubation oven for 10-30 minutes.
  • the slide is air-dried in an inclined position. 18. After applying an antifading reagent to the slide, it can be viewed under an epifluroescence microscope.
  • the sample can be fixed differently efficiently. In the subsequent hybridization process, this leads to a differently efficient penetration of the gene probes into the cells and therefore to different brightnesses when the cells are detected under the epifluorescence microscope. However, the cause correlates with the brightness with the ribosome content of the cells. Therefore e.g. in the FISH analysis, the intensity of the fluorescence is a measure of whether the cells were in a good or poor growth state at the time of sampling. This statement is important for an overall assessment of the microbial state of a sample, especially in medical microbiology, but also in food or environmental microbiology. Consequently, differently efficient fixation of the sample to be examined leads to falsified statements about the state of growth and thus about the overall microbial state of a sample.
  • the preparation of the moist chamber is cumbersome and does not guarantee a horizontal position of the slide. Under certain circumstances, this can lead to a mixing of the solutions located on the various cutouts.
  • the slide must first be rinsed during the washing process and then transferred to another container. Due to this relatively lengthy process, non-specific binding of nucleic acid probe molecules to the cells can take place due to the reduced hybridization temperatures.
  • the object of the present invention is therefore to overcome the disadvantages of the prior art described above and to provide a device or an arrangement and a method with which rapid identification of microorganisms in a sample can be carried out simply and reproducibly.
  • an in situ hybridization arrangement for the specific detection of microorganisms which has a container (1) provided with at least one opening (2); a carrier for the hybridization solution (3); a slide (4); and fastening means (5) for the slide.
  • the arrangement preferably comprises a lid (6) which is suitable for sealing off, in particular for closing the opening of the container in a water and / or airtight manner.
  • the term “leakproof” is to be understood in such a way that the moisture present in the container does not essentially escape from the container in the closed state.
  • the slide is preferably provided with cutouts (9) into which the sample to be examined and, if appropriate, negative or positive samples can be applied separately from one another.
  • the cutouts on the slide are particularly preferably only adjacent in one dimension to further cutouts, e.g. arranged in a row, the recesses can also be offset within the row.
  • the cover comprises the fastening means (5) for the slide.
  • the cover particularly preferably comprises, as fastening means for the specimen slide, a slot (5) into which one end (7) of the specimen slide can be inserted.
  • the slide can of course also engage in fasteners (5) which are part of the container and e.g. in the form of a slit in the bottom of the container, the bottom being in this case opposite the opening of the container.
  • the cover is provided with a component (8) which is stable
  • the position of the lid with the attached slide enables it to be separated from the arrangement without a lid when the slide is in a horizontal position.
  • the lid is particularly preferably designed such that it is possible to stand the lid with the attached slide separately from the arrangement without a lid when the slide is in a horizontal, vertical or lateral position.
  • the horizontal position of the slide is understood to mean that the position of the slide is such that it is possible to apply samples or probes to the slide without flowing.
  • the lateral position of the slide is understood to mean a position rotated by 90 ° with respect to the horizontal position, the slide being rotated by 90 ° in such a way that drops can run off the slide without possibly running into another recess on the slide , provided that the cutouts are only adjacent to one another in one dimension.
  • Invention understood a position rotated by 90 ° with respect to both the horizontal and the lateral position.
  • the container and / or the lid are constructed in such a way that both when the lid is not closed and when the lid is closed, a stable standing position of the arrangement is possible when the specimen slide is in a horizontal or vertical or lateral position.
  • an arrangement is further preferred in which lateral guide rails (10) for the specimen slide are attached to the container for stabilizing the specimen slide in the container or such guide rails (10) are part of the container.
  • the hybridization solution carrier (3) can preferably be removed or used. It is further preferred according to the invention that the carrier for the hybridization solution (3) can be completely introduced into the container. Preferably, however, the carrier for the hybridization solution (3) can also be partially, in particular continuously, introduced into the container (1).
  • lateral guide rails (11) for the hybridization solution carrier (3) are attached to the container (1) for stabilizing the hybridization solution carrier (3) in the container (1) or such guide rails (11) are part of the Container (1).
  • the carrier for the hybridization solution is an integral part of the container, in particular a recess in the container.
  • the carrier for the hybridization solution is a tub (3), in particular a tub, which is provided with cutouts (12) for holding liquid and / or for holding pillows soaked in liquid.
  • the materials for all components of the arrangement, except for the slide preferably comprise plastics, particularly preferably polyethylene and / or polypropylene. Furthermore, the materials for the above-mentioned components of the arrangement can also comprise metals.
  • the slide is preferably made of glass, particularly preferably of glass that corresponds to hydrolytic classes 1 to 4 according to DIN 12111.
  • a method for the specific detection of microorganisms by in situ hybridization comprises the following steps:
  • steps a) to c) and optionally d) are carried out with the in situ hybridization arrangement according to the invention.
  • fixation and / or drying steps are preferably carried out on the object carrier.
  • the final drying of the slide is carried out in the lateral position of the slide and / or the incubation with the slide in a horizontal position and / or washing with the slide in a vertical position.
  • a mixture of a hybridization solution and a nucleic acid probe molecule solution is applied to the slide in step b).
  • this drip container is a disposable drip container or a drip container for repeated use.
  • the hybridization solution which is required for the moist chamber can be introduced into the arrangement according to the invention in the method according to the invention via cushions located in the carrier for the hybridization solution and soaked with hybridization solution.
  • the nucleic acid probe molecule used in step b) is preferably complementary to a chromosomal or episomal DNA, an mRNA or rRNA of a microorganism to be detected.
  • the nucleic acid probe molecule is covalently linked to a detectable marker.
  • This detectable marker is preferably selected from the group of the following markers:
  • Fluorescent marker Fluorescent marker, chemiluminescent marker, radioactive marker, - enzymatically active group,
  • the microorganism is preferably a single-cell microorganism.
  • the microorganism is particularly preferably a yeast, a bacterium, an alga or a fungus.
  • the microorganism is a wastewater bacterium.
  • the sample is an environmental sample and is taken from water, soil or air; or a food sample, in particular a sample from milk or milk products, drinking water, beverages, baked goods or meat products; or a medical sample, in particular a sample obtained from tissue, secretions or stool; or a sample from waste water, in particular a sample obtained from activated sludge, digested sludge or anaerobic sludge; or a sample obtained from a biofilm, in particular a sample in which the biofilm is obtained from an industrial plant, in which wastewater treatment was formed or is a natural biofilm; or a sample taken from a pharmaceutical or cosmetic product.
  • kits for the specific detection of microorganisms by in situ hybridization which contains at least one nucleic acid probe molecule for the specific detection of a microorganism; at least one hybridization solution; if applicable
  • Nucleic acid probe molecule for performing a negative control optionally a nucleic acid probe molecule for performing a positive control; optionally a washing solution, optionally one Fixing solution, optionally an antifading reagent and an in situ hybridization arrangement according to the invention.
  • the nucleic acid probe molecule in the kit according to the invention is preferably complementary to a cliromosomal or episomal DNA, an mRNA or rRNA of a microorganism to be detected.
  • the nucleic acid probe molecule in the kit according to the invention is preferably covalently linked to a detectable marker.
  • the detectable marker is particularly preferably selected from the group consisting of fluorescent markers, chemiluminescent markers, radioactive markers, enzymatically active groups, haptens and nucleic acids detectable by hybridization.
  • Another object of the present invention is the use of the in situ hybridization arrangement according to the invention for the specific detection of microorganisms by in situ hybridization.
  • the present invention furthermore relates to the use of the kit according to the invention in the method according to the invention.
  • the arrangement according to the invention comprises a container provided with at least one opening, which is also referred to below as a chamber; a carrier for the hybridization solution, in particular a tub, which can be completely introduced into the container or the chamber or is part of this container; as well as a mounting option for a slide.
  • the arrangement further comprises a slide which can be inserted into the chamber for in situ hybridization.
  • the arrangement according to the invention preferably further comprises a lid which is suitable for sealing off, in particular for watertight and / or airtight, opening of the container.
  • the object holder is preferably attached to this cover, particularly preferably inserted.
  • the object holder is firmly and securely in the lid, but can be pulled out of the lid again for final evaluation by hand and without great effort.
  • the fixing or fastening of the lid makes it possible to carry out the washing process safely even when the arrangement according to the invention is in a vertical position.
  • the cover is provided with a component or the cover comprises a component which enables the cover with a fixed slide to stand stable and separate from the arrangement with the slide in a horizontal or vertical position.
  • This stability of the lid which was achieved according to the invention, for example by attaching a support leg, makes it possible to keep the slide in a horizontal position during all reactions taking place.
  • the slide is preferably attached to the lid of the in situ hybridization arrangement (see Figure 4).
  • the object holder can remain connected to the lid during the entire hybridization method according to the invention.
  • all preparatory procedures such as washing, fixing and the like are thereby in the same Reaction chamber possible.
  • Providing the lid with a component that enables a stable position of the lid separately from the arrangement in the horizontal or vertical position of the slide has the essential advantage that the slide can be left in the lid even when the samples and probes are applied and at the same time a level and secure stand is guaranteed when applying the samples.
  • the component or the supporting leg of the lid enables the individual reactions to be carried out to bring about the hybridization of nucleic acid probes with cells when the slide is attached to the lid on this slide. Furthermore, with such a component provided cover all drying processes can also be carried out outside the chamber. The level and secure position of the lid with the attached slide can prevent the samples from flowing on the slide.
  • the container and / or the lid are constructed in such a way that, when the lid is closed, the arrangement is stable when the slide is in a horizontal position.
  • the horizontal storage of the slide, in particular during steps a) and b) of the method, is thus ensured by the construction of the contact surfaces of the components of the in situ hybridization arrangement according to the invention.
  • the use of a tub as a carrier for the hybridization solution enables the hybridization solution which is required for the moist chamber to be placed safely and cleanly in the arrangement according to the invention.
  • the tub preferably has small cutouts for holding liquid (see Figure 5). It is also particularly preferred that the tub is initially only partially inserted into the chamber, so that the hybridization solution is responsible for the guarantee a moist chamber is required, into which the tub can be placed. The tub is then preferably pushed all the way into the chamber (see Figure 6).
  • cellulose can be used as a carrier for the hybridization solution.
  • Another alternative for introducing the hybridization solution into the in situ hybridization arrangement according to the invention is that
  • hybridization solution into the reactor using disposable cushions that are located in the tub.
  • the disposable pillows are sealed with a freshness seal, which is torn off as soon as the tub is in the chamber, and the hybridization solution can then evaporate in the chamber.
  • Both the incubation and the washing process preferably take place in the in situ hybridization arrangement according to the invention.
  • microorganisms are preferably not first fixed in a reaction vessel and then immobilized, as is usually the case, but rather the fixing and / or optionally the drying take place directly on the slide.
  • fixation on the slide avoids cell loss and is much easier to use and less complicated to carry out.
  • fixation on the slide enables the fixation step and the dehydration series to be combined in one work step.
  • the hybridization and addition of the nucleic acid probe molecules is preferably not carried out by, as is customary, first a defined amount of a hybridization solution and then a defined amount of one
  • Probe solution is pipetted into a specimen slide recess by means of a pipette, but rather by applying a mixture of a hybridization and a nucleotide probe molecule solution to the specimen slide.
  • Nucleic acid probe molecule solution enables a faster and flawless procedure with the same stability of the nucleic acid probe molecules.
  • the above-mentioned mixture is preferably applied dropwise through a dropping vessel by applying slight pressure.
  • the drip vessel can either be intended for repeated use and contain several drops of the mixed solution from the hybridization and the probe solution, or alternatively it can be a small one-time drip vessel which contains the required amount of reagents while taking into account a dead volume.
  • drip containers saves the use of expensive pipettes and also makes handling and dosing easier.
  • the slide which is preferably attached to the lid, is then inserted into the chamber (see Figure 7).
  • Lateral guide lines which are preferably installed in the chamber, ensure problem-free insertion and further fixation or stabilization of the slide in the chamber (see Figure 8).
  • the chamber and / or the cover is preferably constructed in such a way that a stable horizontal, vertical and lateral standing position is ensured.
  • the subsequent incubation is preferably carried out in the horizontal position of the slide.
  • the subsequent washing of the slide is preferably carried out according to the invention in the chamber and particularly preferably in the vertical position of the slide.
  • the lid is preferably constructed so that it can be placed on the side. This is particularly advantageous because the drops can run off the slide without running into another recess in the slide.
  • FIG. 9 shows the entire structure of a preferred embodiment of the in situ hybridization arrangement according to the invention, comprising the lid, chamber, tub and supplied slide.
  • Figures 10 to 12 show the dimensions of the lid, pan and chamber.
  • fixing microorganisms is understood to mean a treatment with which the cell envelope of the microorganisms is made permeable to nucleic acid probes.
  • the nucleic acid probes which consist of an oligonucleotide and a marker attached to it, can then penetrate the cell envelope and bind to the target sequence corresponding to the nucleic acid probe inside the cell.
  • the bond is as an education of
  • the envelope can be a lipid envelope that surrounds a virus, around the cell wall of bacteria or around the cell membrane of one cell Eukaryotes. Ethanol is usually used for fixation. If these measures cannot be used to make the cell wall penetrable by nucleic acid probes, the person skilled in the art is sufficiently aware of further measures which lead to the same result. These include, for example, a low-percentage paraformaldehyde solution or a dilute formaldehyde solution, methanol, mixtures of alcohols, enzymatic treatments or the like.
  • the nucleic acid probe in the sense of the invention can be a DNA or RNA probe which will generally comprise between 12 and 1000 nucleotides, preferably between 12 and 50, particularly preferably between 17 and 25 nucleotides.
  • the nucleic acid probes are selected on the basis of whether a complementary sequence is present in the microorganism to be detected. By selecting a defined sequence, a bacterial species, a bacterial genus or an entire bacterial group can be detected. Complementarity must be given for a probe of 12 to 15 nucleotides in length over 100% of the sequence. With oligonucleotides with more than 15 nucleotides, one or more mismatching sites are allowed. Compliance with stringent hybridization conditions ensures that the nucleic acid probe molecule actually hybridizes with the target sequence. Moderate conditions in the sense of the invention are e.g. 0% formamide in one
  • Hybridization solution as described in Example 1.
  • Stringent conditions in the sense of the invention are, for example, 20-80% formamide in the hybridization solution.
  • the duration of the hybridization is usually between 10 minutes and 12 hours; hybridization is preferably carried out for about 2 hours.
  • the hybridization temperature is preferably between 44 ° C. and 48 ° C., particularly preferably 46 ° C., the parameter of the hybridization temperature, like that Concentration of salts and detergents in the hybridization solution depending on the probe or probes, in particular their length (s) and the degree of complementarity to the target sequence in the cell to be detected can be optimized.
  • the person skilled in the art is familiar with the relevant calculations here.
  • a typical hybridization solution has a salt concentration of 0.1 M to 1.5 M, preferably 0.9 M, the salt preferably being sodium chloride.
  • the hybridization solution usually comprises a detergent, e.g.
  • Tris / HCl in a concentration range of 0.001-0.1 M, preferably in a concentration of 0.02 M.
  • the pH of Tris / HCl is usually between 6 and 10, with a pH of about 8.0 being preferred.
  • the hybridization solution may further contain between 0% and 80% formamide, depending on what degree of stringency is desired or needed.
  • the nucleic acid probe should be present in the hybridization solution in an amount of from 15 ng to 1000 ng, this amount being contained in a volume of hybridization solution between 8 ⁇ l and 100 ⁇ l, preferably in 40 ⁇ l.
  • the probe concentration is particularly preferably 111 ng / 40 ⁇ l hybridization solution.
  • the non-hybridized and excess probe molecules should be removed, which is usually done using a conventional washing solution or a conventional washing buffer.
  • This wash solution can, if desired, 0.001-0.1% of a detergent such as SDS, one Concentration of 0.01% is preferred, as well as Tris / HCl in a concentration of 0.001-0.1 M, preferably 0.02 M, the pH of Tris / HCl in the range from 6.0 to 10, 0, preferably 8.0.
  • a detergent may be included, but is not essential.
  • the washing solution usually also contains NaCl, the concentration depending on the stringency required being from 0.003 M to 0.9 M, preferably from 0.01 M to 0.9 M.
  • the washing solution can contain EDTA, the concentration preferably being 0.005 M.
  • the washing solution can also contain preservatives known to the person skilled in the art in suitable amounts.
  • the “washing off” of the unbound probe molecules is usually carried out at a temperature in the range from 44 ° C. to 52 ° C., preferably from 44 ° C. to 50 ° C. and particularly preferably at 46 ° C. for a period of 10-40 minutes, preferably for 15 minutes.
  • the selection of the respective nucleic acid probes depends on the microorganism to be detected.
  • the nucleic acid probe can be complementary to a cliromosomal or episomal DNA, but also to an mRNA or rRNA of the microorganism to be detected. It is advantageous to choose a nucleic acid probe that is complementary to an area that is present in the number of copies of more than 1 in the microorganism to be detected.
  • the sequence to be detected is preferably 500-100,000 times per cell, particularly preferably 1000-50,000 times.
  • the rRNA is preferably used as the target site, since the ribosomes in the cell, as sites for protein biosynthesis, are present thousands of times in each active cell.
  • the nucleic acid probe is incubated with the microorganism fixed in the abovementioned sense, so as to prevent the To allow nucleic acid probe molecules in the microorganism and the hybridization of nucleic acid probe molecules with the nucleic acids of the microorganism. Then the non-hybridized
  • Nucleic acid probe molecules removed by usual washing steps.
  • the specifically hybridized nucleic acid probe molecules can then be detected in the respective cells.
  • nucleic acid probe molecule used according to the invention is detectable. This traceability can e.g. by covalent connection of the nucleic acid probe molecule used according to the invention.
  • Nuclear acid probe molecule can be ensured with a detectable marker.
  • Fluorescent groups such as e.g. CY2, CY3, CY5, FITC, FLUOS, TRITC or FLUOS-PRIME are used, all of which are well known to those skilled in the art. Examples of fluorescent groups are given in Table 1 below:
  • TRITC Teframethylrhodamine-5.6 isothiocyanate (Isomer G. Molecular Probes
  • chemiluminescent groups or radioactive labels for example 35 S, 32 P, 33 P, 125 J
  • detectability can also be provided by coupling the nucleic acid probe molecule with an enzymatically active molecule, for example alkaline phosphatase, acid phosphatase, peroxidase, horseradish peroxidase, ⁇ -D-galactosidase or glucose oxidase.
  • an enzymatically active molecule for example alkaline phosphatase, acid phosphatase, peroxidase, horseradish peroxidase, ⁇ -D-galactosidase or glucose oxidase.
  • chromogens are known for each of these enzymes, which can be converted instead of the natural substrate and can be converted into either colored or fluorescent products. Examples of such chromogens are given in Table 2 below. TABLE 2
  • nucleic acid probe molecules in such a way that a further nucleic acid sequence suitable for hybridization is present at their 5 'or 3' end.
  • This nucleic acid sequence in turn comprises approximately 12 to 1000, preferably 15-50 nucleotides.
  • This second nucleic acid region can in turn be detected by an oligonucleotide probe which can be detected by one of the means mentioned above.
  • Another possibility is to couple the detectable nucleic acid probe molecules with a hapten. After the nucleic acid probe molecules have been detached from the target nucleic acid, the nucleic acid probe molecules, which are now present separately, can be brought into contact with detectable antibodies which recognize the hapten.
  • detectable antibodies which recognize the hapten.
  • a well-known example of such a hapten is digoxigenin or its derivatives.
  • the person skilled in the art knows many other possibilities for detecting and quantifying an oligonucleotide used for hybridization.
  • the multitude of possible markings also enables the simultaneous detection of two or more overlapping or non-overlapping populations. For example, by using two or more different fluorescent markers, several bacterial populations can be detected (R. Amann, J. Snaidr, M. Wagner, W. Ludwig, and K.-H. Schleifer. 1996. In situ visualization of high genetic diversity in a natural microbial community. J. Bacteriol. 178: 12, 3496-3500).
  • the evaluation depends on the type of marking of the probe used. In the context of the present invention, the evaluation can advantageously be carried out using a light microscope, epifluorescence microscope, chemiluminometer, fluorometer and the like.
  • the microorganism to be detected by means of the method according to the invention can be a prokaryotic or eukaryotic microorganism. In most cases, it will be desirable to detect unicellular microorganisms. These unicellular microorganisms can also be present in larger aggregates, so-called filaments. Relevant microorganisms are above all yeasts, algae, bacteria or fungi.
  • the microorganisms are bacteria which occur in the waste water from waste water treatment plants.
  • the method according to the invention can be used in a variety of ways.
  • Environmental samples can be examined for the presence of microorganisms.
  • these samples can be taken from air, water or from the ground.
  • the food samples are made from milk or milk products (yogurt, cheese, curd cheese, butter, buttermilk), drinking water, cupboards (lemonades, beer, juices), baked goods or
  • the method according to the invention can also be used to examine medical samples. It is suitable for the examination of tissue samples, e.g. biopsy material from the lungs, tumor or inflammatory tissue, from secretions such as sweat, saliva, sperm and discharge from the nose, urethra or vagina as well as for urine or stool samples.
  • tissue samples e.g. biopsy material from the lungs, tumor or inflammatory tissue, from secretions such as sweat, saliva, sperm and discharge from the nose, urethra or vagina as well as for urine or stool samples.
  • Another area of application for the present method is the investigation of waste water, e.g. Activated sludge, digested sludge or anaerobic sludge.
  • waste water e.g. Activated sludge, digested sludge or anaerobic sludge.
  • it is suitable to analyze biofilms in industrial plants, as well as to investigate naturally forming biofilms or biofilms that form during waste water purification.
  • Examination of pharmaceutical and cosmetic products e.g. Ointments, creams, tinctures, juices etc. are possible with the method according to the invention.
  • a kit for carrying out the method for the detection of microorganisms in a sample is provided in a further aspect of the present invention.
  • the content of such a kit depends essentially on the nature of the microorganism to be detected. As the most important constituent, it comprises one or more nucleic acid probes specific for the microorganism to be detected in each case and preferably further nucleic acid probes with which a negative or positive control can be carried out. In addition, it preferably comprises a hybridization solution and a washing solution. The choice of hybridization solution depends primarily on the length of the nucleic acid probes used.
  • hybridization conditions are eg in steel & Amann (1991) in Stackebrand u. Goodfellow (ed.), Nucleic Acid Techniques in Bacterial Systematics; John Wiley & Sons Ltd., Chichester, UK.
  • the kit according to the invention comprises at least one nucleic acid probe molecule for the specific detection of a microorganism; at least one hybridization solution; optionally a nucleic acid probe molecule for performing a negative control; optionally a nucleic acid probe molecule for performing a positive control; optionally a washing solution; optionally a fixing solution, optionally an antifading reagent; and the in situ hybridization arrangement according to the invention, the following steps being able to be carried out in the arrangement or in parts of the arrangement:
  • the kit contains specific probes for the detection of bacteria which occur in the wastewater of wastewater treatment plants.
  • In situ hybridization can be established in practice by the method according to the invention.
  • VIT method in the in situ hybridization arrangement according to the invention, hereinafter also referred to as "VIT reactor”, is used for the qualitative analysis of bacteria which are in Waste water samples occur. The identification takes place in a few hours.
  • the bacteria are hybridized with fluorescence-labeled oligonucleotide probes using this method and can then be detected on the slide using an epifluorescence microscope.
  • Drying cabinet preheated to 46 ° C bottle for preparing and heating the washing solution.
  • Measuring cylinder for preparing the washing solution.
  • Thermometer alarm clock
  • VIT solution solution with specific nucleic acid probe molecules.
  • Negative Confrol solution for negative control
  • Positive Confrol Solution for positive control
  • washing solution 25 ml of washing solution are required per NIT reactor.
  • Solution D is diluted ten times with distilled water.
  • Table 3 shows ner thinning examples.
  • Illustration 1
  • Figure 3 Top view of the components of a special embodiment of the in situ hybridization arrangement according to the invention: container (1), tub (3), object holder (4), lid (6) with support leg (8) (from left to right)
  • Figure 4 Schematic representation of a particularly preferred embodiment of the
  • FIG. 5 Schematic representation of a preferred embodiment of the in situ hybridization arrangement according to the invention.
  • the tub (3) has small recesses for holding liquid and is initially only partially inserted into the chamber (1) in order to be able to add the hybridization solution, which is required for a moist chamber, to the tub (3).
  • Figure 12 Scale drawing of a preferred embodiment of the container (1) or the chamber (1).

Landscapes

  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Organic Chemistry (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Analytical Chemistry (AREA)
  • Zoology (AREA)
  • Health & Medical Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Wood Science & Technology (AREA)
  • Microbiology (AREA)
  • Physics & Mathematics (AREA)
  • Molecular Biology (AREA)
  • Immunology (AREA)
  • Biotechnology (AREA)
  • Biophysics (AREA)
  • Biochemistry (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • General Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
  • Apparatus Associated With Microorganisms And Enzymes (AREA)

Abstract

L'invention concerne un système d'hybridation <i>in situ</i> de mise en évidence spécifique de micro-organismes, un procédé de mise en évidence spécifique de micro-organismes par hybridation <i>in situ</i>, ainsi qu'un kit permettant d'identifier et de visualiser des micro-organismes dans un échantillon. L'invention concerne en particulier un système d'hybridation <i>in situ</i> de mise en évidence spécifique de micro-organismes, qui comprend : un récipient (1) muni d'au moins une ouverture (2) ; un support pour la solution d'hybridation (3) ; un porte-objet (4) et un élément de fixation (5) pour le porte-objet.
EP01991823A 2000-12-11 2001-12-11 Systeme d'hybridation in situ de mise en evidence specifique de micro-organismes Expired - Lifetime EP1341897B1 (fr)

Applications Claiming Priority (3)

Application Number Priority Date Filing Date Title
DE10061655 2000-12-11
DE10061655A DE10061655A1 (de) 2000-12-11 2000-12-11 In situ-Hybridisierungs-Anordnung zum spezifischen Nachweis von Mikroorganismen
PCT/EP2001/014543 WO2002048398A2 (fr) 2000-12-11 2001-12-11 Systeme d'hybridation in situ de mise en evidence specifique de micro-organismes

Publications (2)

Publication Number Publication Date
EP1341897A2 true EP1341897A2 (fr) 2003-09-10
EP1341897B1 EP1341897B1 (fr) 2005-06-29

Family

ID=7666693

Family Applications (1)

Application Number Title Priority Date Filing Date
EP01991823A Expired - Lifetime EP1341897B1 (fr) 2000-12-11 2001-12-11 Systeme d'hybridation in situ de mise en evidence specifique de micro-organismes

Country Status (8)

Country Link
US (1) US20040038270A1 (fr)
EP (1) EP1341897B1 (fr)
JP (1) JP2004523223A (fr)
AT (1) ATE298786T1 (fr)
AU (1) AU2002231689A1 (fr)
CA (1) CA2430894A1 (fr)
DE (2) DE10061655A1 (fr)
WO (1) WO2002048398A2 (fr)

Families Citing this family (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
DE10323197B4 (de) * 2003-05-22 2008-10-02 Clondiag Chip Technologies Gmbh Vorrichtung zur Halterung und Detektion von Substanzbibliotheken
US6995020B2 (en) * 2003-07-21 2006-02-07 Aureon Laboratories, Inc. Methods and compositions for the preparation and use of fixed-treated cell-lines and tissue in fluorescence in situ hybridization
US8062897B2 (en) 2003-07-21 2011-11-22 Aureon Laboratories, Inc. Diagnostic histopathology using multiplex gene expression FISH
JP4785449B2 (ja) * 2005-07-21 2011-10-05 公益財団法人函館地域産業振興財団 培養併用蛍光インサイチューハイブリダイゼーション法による食品の微生物検査法
US8481302B2 (en) * 2008-11-03 2013-07-09 General Electric Company Total bacteria monitoring system
ES2387903B1 (es) * 2010-07-16 2013-08-13 María Albiñana Blanco Procedimiento y kit para verificar, en una muestra de semen humano o animal, la existencia de espermatozoides fecundantes.
DE102011004449B4 (de) * 2011-02-21 2019-01-24 Fraunhofer-Gesellschaft zur Förderung der angewandten Forschung e.V. Biopsieplättchen, Einbettkassette und Diagnosevorrichtung
CN106442020B (zh) * 2016-11-18 2022-05-24 何耀 一种尿液取样检测装置
CN110672811A (zh) * 2019-10-21 2020-01-10 远大可建科技有限公司 一种芯板内腔检测装置及其方法

Family Cites Families (17)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US3618265A (en) * 1969-01-08 1971-11-09 Remington Arms Co Inc Finishing machine for metal surfaces
US3616265A (en) * 1969-08-07 1971-10-26 Smith Kline French Lab Device for making a culture of microorganisms
FR2525233B1 (fr) * 1982-04-14 1985-12-06 Lille Inst Pasteur Dispositif de culture microbiologique
US4640895A (en) * 1982-10-15 1987-02-03 Gibco Division, The Mogul Corporation Biphasic media culture apparatus
DE8309876U1 (de) * 1983-04-02 1983-12-22 Biotest-Serum-Institut Gmbh, 6000 Frankfurt Zweiphasige blutkulturflasche
IT1205100B (it) * 1987-06-25 1989-03-10 Sta Te Spa Dispositivo per culture microbiologiche
US4886741A (en) * 1987-12-09 1989-12-12 Microprobe Corporation Use of volume exclusion agents for the enhancement of in situ hybridization
US5192503A (en) * 1990-05-23 1993-03-09 Mcgrath Charles M Probe clip in situ assay apparatus
US5380492A (en) * 1990-12-18 1995-01-10 Seymour; Eugene H. Sampling device and sample adequacy system
EP0497464A1 (fr) * 1991-01-31 1992-08-05 Amoco Corporation Diagnose rapide de microbes par hybridisation in situ en suspension aqueuse
US5128476A (en) * 1991-02-20 1992-07-07 The Midland Certified Reagent Company Biotinylated oligonucleotides and reagents for preparing the same
AU2941092A (en) * 1991-11-15 1993-06-15 Nunc A/S A cell cultivating device
DE4409705A1 (de) * 1994-03-22 1995-09-28 Boehringer Mannheim Gmbh Vorrichtung zur Bearbeitung von Nukleinsäuren in Präparationen
US6022689A (en) * 1995-04-07 2000-02-08 University Of New Mexico Situ hybridization slide processes
US5750340A (en) * 1995-04-07 1998-05-12 University Of New Mexico In situ hybridization solution and process
US6007994A (en) * 1995-12-22 1999-12-28 Yale University Multiparametric fluorescence in situ hybridization
WO2000065093A2 (fr) * 1999-04-22 2000-11-02 Science And Technology Corporation Procedes d'hybridation in situ permettant de reduire l'occurrence de faux resultats positifs et de cibler des micro-organismes multiples

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
See references of WO0248398A2 *

Also Published As

Publication number Publication date
ATE298786T1 (de) 2005-07-15
CA2430894A1 (fr) 2002-06-20
EP1341897B1 (fr) 2005-06-29
JP2004523223A (ja) 2004-08-05
DE10061655A1 (de) 2002-06-20
DE50106653D1 (de) 2005-08-04
WO2002048398A2 (fr) 2002-06-20
AU2002231689A1 (en) 2002-06-24
US20040038270A1 (en) 2004-02-26
WO2002048398A3 (fr) 2003-05-15

Similar Documents

Publication Publication Date Title
EP1198597A1 (fr) Procede permettant de detecter des organismes de fa on specifique a l&#39;espece
EP1177319B1 (fr) Procede de detection de micro-organismes dans un echantillon
EP1341897B1 (fr) Systeme d&#39;hybridation in situ de mise en evidence specifique de micro-organismes
WO2018099500A1 (fr) Préparation d&#39;échantillons microbiens vivants et de microorganismes pour effectuer ensuite une mesure et une évaluation par spectrométrie de masse
CN103476945A (zh) 使用基于分子核酸的技术确定细胞活性的改进方法
DE10344057B3 (de) Verfahren zum spezifischen Schnellnachweis getränkeschädlicher Mikroorganismen
EP1523579A2 (fr) Oligonucleotides pour la detection de micro-organismes
DE19934510B4 (de) Verfahren zum Nachweis von Mikroorganismen
DE10129410A1 (de) Verfahren zum spezifischen Schnellnachweis bierschädlicher Bakterien
EP1490507B1 (fr) Procede de detection de micro-organismes par une hybridation in situ et une cytometrie de flux
DE202012005258U1 (de) Mittel zur schnellen quantitativen und qualitativen Bestimmung von Schmimmelpilzen und Hefen
US20070243532A1 (en) System and Method of Detecting a Microorganism
DE10010614A1 (de) Oligonukleotidsonden zum art- und/oder gattungsspezifischen Nachweis von das Pflanzenwachstum fördernden Bakterien
DE10022304B4 (de) Verfahren zur Identifizierung, Quantifizierung und Visualisierung von Mikroorganismen
JP2002500020A (ja) 嫌気性細菌の毒性化合物による阻止および殺害を迅速に評価する方法
EP1335991B1 (fr) Procede pour detecter des protozoaires du genre naegleria
AT512416B1 (de) Anordnung und verfahren zum nachweis von mikroorganismen in einem kulturgefäss
DE102020103961B4 (de) Verfahren zum Nachweis von Mikroorganismen, korrespondierende Verwendung und Probenträger
DE10121505A1 (de) Verfahren zum Nachweis von Gram-positiven Bakterien
DE102008002978B4 (de) Verfahren zur Differenzierung von verschiedenen Spezies der Gattung Methylobacterium
EP1490518B1 (fr) Procede de detection et de differenciation du complexe mycobacterium tuberculosis
DE10307732A1 (de) Oligonukleotide zum Nachweis von Mikroorganismen
DE10160666A1 (de) Verfahren zum spezifischen Schnellnachweis von Trinkwasser relevanten Bakterien
EP2877595A1 (fr) Procédé de différenciation entre cellules vivantes et mortes

Legal Events

Date Code Title Description
PUAI Public reference made under article 153(3) epc to a published international application that has entered the european phase

Free format text: ORIGINAL CODE: 0009012

17P Request for examination filed

Effective date: 20030707

AK Designated contracting states

Kind code of ref document: A2

Designated state(s): AT BE CH CY DE DK ES FI FR GB GR IE IT LI LU MC NL PT SE TR

AX Request for extension of the european patent

Extension state: AL LT LV MK RO SI

GRAP Despatch of communication of intention to grant a patent

Free format text: ORIGINAL CODE: EPIDOSNIGR1

GRAS Grant fee paid

Free format text: ORIGINAL CODE: EPIDOSNIGR3

GRAA (expected) grant

Free format text: ORIGINAL CODE: 0009210

AK Designated contracting states

Kind code of ref document: B1

Designated state(s): AT BE CH CY DE DK ES FI FR GB GR IE IT LI LU MC NL PT SE TR

PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: IT

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT;WARNING: LAPSES OF ITALIAN PATENTS WITH EFFECTIVE DATE BEFORE 2007 MAY HAVE OCCURRED AT ANY TIME BEFORE 2007. THE CORRECT EFFECTIVE DATE MAY BE DIFFERENT FROM THE ONE RECORDED.

Effective date: 20050629

Ref country code: TR

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20050629

Ref country code: NL

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20050629

Ref country code: FI

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20050629

REG Reference to a national code

Ref country code: GB

Ref legal event code: FG4D

Free format text: NOT ENGLISH

REG Reference to a national code

Ref country code: CH

Ref legal event code: EP

REF Corresponds to:

Ref document number: 50106653

Country of ref document: DE

Date of ref document: 20050804

Kind code of ref document: P

REG Reference to a national code

Ref country code: IE

Ref legal event code: FG4D

Free format text: LANGUAGE OF EP DOCUMENT: GERMAN

PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: SE

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20050929

Ref country code: DK

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20050929

Ref country code: GR

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20050929

PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: ES

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20051010

GBT Gb: translation of ep patent filed (gb section 77(6)(a)/1977)

Effective date: 20050920

REG Reference to a national code

Ref country code: CH

Ref legal event code: NV

Representative=s name: ISLER & PEDRAZZINI AG

NLV1 Nl: lapsed or annulled due to failure to fulfill the requirements of art. 29p and 29m of the patents act
PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: PT

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20051207

PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: CY

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20051211

PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: LU

Free format text: LAPSE BECAUSE OF NON-PAYMENT OF DUE FEES

Effective date: 20051231

Ref country code: MC

Free format text: LAPSE BECAUSE OF NON-PAYMENT OF DUE FEES

Effective date: 20051231

ET Fr: translation filed
PLBE No opposition filed within time limit

Free format text: ORIGINAL CODE: 0009261

STAA Information on the status of an ep patent application or granted ep patent

Free format text: STATUS: NO OPPOSITION FILED WITHIN TIME LIMIT

26N No opposition filed

Effective date: 20060330

REG Reference to a national code

Ref country code: CH

Ref legal event code: PCAR

Free format text: ISLER & PEDRAZZINI AG;POSTFACH 1772;8027 ZUERICH (CH)

PGFP Annual fee paid to national office [announced via postgrant information from national office to epo]

Ref country code: CH

Payment date: 20081222

Year of fee payment: 8

Ref country code: IE

Payment date: 20081218

Year of fee payment: 8

PGFP Annual fee paid to national office [announced via postgrant information from national office to epo]

Ref country code: FR

Payment date: 20081216

Year of fee payment: 8

PGFP Annual fee paid to national office [announced via postgrant information from national office to epo]

Ref country code: GB

Payment date: 20081219

Year of fee payment: 8

PGFP Annual fee paid to national office [announced via postgrant information from national office to epo]

Ref country code: BE

Payment date: 20090107

Year of fee payment: 8

BERE Be: lapsed

Owner name: *VERMICON A.G.

Effective date: 20091231

REG Reference to a national code

Ref country code: CH

Ref legal event code: PL

GBPC Gb: european patent ceased through non-payment of renewal fee

Effective date: 20091211

REG Reference to a national code

Ref country code: FR

Ref legal event code: ST

Effective date: 20100831

REG Reference to a national code

Ref country code: IE

Ref legal event code: MM4A

PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: IE

Free format text: LAPSE BECAUSE OF NON-PAYMENT OF DUE FEES

Effective date: 20091211

Ref country code: BE

Free format text: LAPSE BECAUSE OF NON-PAYMENT OF DUE FEES

Effective date: 20091231

Ref country code: CH

Free format text: LAPSE BECAUSE OF NON-PAYMENT OF DUE FEES

Effective date: 20091231

Ref country code: FR

Free format text: LAPSE BECAUSE OF NON-PAYMENT OF DUE FEES

Effective date: 20091231

Ref country code: LI

Free format text: LAPSE BECAUSE OF NON-PAYMENT OF DUE FEES

Effective date: 20091231

PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: GB

Free format text: LAPSE BECAUSE OF NON-PAYMENT OF DUE FEES

Effective date: 20091211

PGFP Annual fee paid to national office [announced via postgrant information from national office to epo]

Ref country code: AT

Payment date: 20101220

Year of fee payment: 10

REG Reference to a national code

Ref country code: AT

Ref legal event code: MM01

Ref document number: 298786

Country of ref document: AT

Kind code of ref document: T

Effective date: 20121211

PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: AT

Free format text: LAPSE BECAUSE OF NON-PAYMENT OF DUE FEES

Effective date: 20121211

PGFP Annual fee paid to national office [announced via postgrant information from national office to epo]

Ref country code: DE

Payment date: 20131217

Year of fee payment: 13

REG Reference to a national code

Ref country code: DE

Ref legal event code: R119

Ref document number: 50106653

Country of ref document: DE

PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: DE

Free format text: LAPSE BECAUSE OF NON-PAYMENT OF DUE FEES

Effective date: 20150701